JPS63171343A - Reagent kit used for testing fluorescence analyzer - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

(Industrial Field of Application) Many commercially available instruments used for particle or cell analysis are used to analyze the properties of particles, particularly biological particles or cells, during analysis or when particles move in flowing water or in suspension. It can be determined with techniques in which the particles remain relatively stable during transport. A typical flow cytometer, also known as a flow cytometer, is useful for characterizing or detecting particles in motion. In a typical flow cytometry instrument, cells or other biological particles are placed in a liquid Flown through running water, each particle is passed through a receptor site, preferably at once, and various signals are sent to the particle, including electrical, acoustic and radioactive, to measure the physical or chemical properties of the particle. Flow cytometers typically rely on optical signals to analyze particles passing through the instrument, although they can be detected in relation to properties. In many cases, whether an analytical instrument is used for particle analysis in either static or dynamic conditions, a preliminary set-up step is usually required to prepare the instrument for use. Since cells or other biological particles are very small and the signal detected for them is often low, it is advisable to carry out appropriate calibration of the instrument before performing the analysis to ensure the reliability of the test results. desirable. PRIOR ART Current techniques for calibrating biological particles or instruments useful for im analysis are highly dependent on the skill of the operator. In other words, tester judgment and experience are required to adjust controls during the testing procedure, determine appropriate thresholds, and maintain multiple standards for each particular test being performed. Although an experienced operator may be very good at calibrating an instrument before use, it requires a great deal of feeling and reflection on the part of such an operator to feel that an adequate qualification has been achieved. For example, when arranging the various optical elements of an instrument such as a flow cytometer, the certifying person typically looks at a screen or CRT, and the instrument is calibrated by visual judgment along with the adjustment of various controls. judge things. Of course, when implementing typical visual verification techniques, there can be compromises in the reproducibility of test results. Flow cytometers and other biological particle analysis instruments typically assay with particles similar to the type of particles or cells being analyzed. Assay particles should therefore be selected to have similar properties to the particles being investigated, such as size, volume, surface properties, granularity, and color characteristics (e.g., 11 cell colorants, dyes, immunofluorescent labels, etc.). . Past and current assay procedures for flow cytometry instruments include the use of chicken red blood cell cells for the assay step; although chicken red blood cells are reliable in some aspects of the assay procedure, they still have some light-related properties in the spectrum. In addition to biological samples for assay purposes, which are not completely satisfactory due to their scarcity, microscopic spherical beads are useful in the assay of cell analysis instruments. The use of beads made of latex material has been described for the verification of particle counting instruments. Assay beads specifically used for calibrating flow cytometry instruments are manufactured by Flow Cytometry Standards Corporation (Flow Cy
tometryS standards Corpora
t 1on), Research Triangle l Repark (Re
search Triangle Park), North Carolina. (Problem to be Solved by the Invention) Even if the effectiveness of assay beads improves the assay technique for particle analysis instruments, it still takes a considerable amount of operator intuition to determine whether the instrument has been properly assayed. is necessary. Therefore, there remains a need and desire to establish assay procedures that not only provide reproducible test results, but also eliminate the deliberation and guesswork associated with currently utilized assay techniques. This is aimed at realizing such improvements. (Means for Solving the Problems) The method of the present invention is a method for calibrating an instrument for obtaining at least one type of light-related signal from particles under analysis. It consists of directing an incident light beam onto a test particle having one or more known characteristics associated with the particle. Both optical and noise signals from the assay particles are detected. Preferably, optical signals are detected from one type of particle, such as fluorescent particles in the assay mixture, and noise signals are detected from the other type of particles, such as non-fluorescent particles, in the assay mixture. The ratio of the detected optical signal to the noise signal is measured and this ratio is recorded. The measured ratio is compared with a previously determined ratio (hereinafter referred to as the predetermined ratio) representing a threshold of the performance limit of the equipment. If the predetermined ratio is not reached, the instrument operation is adjusted while the calibrated particles are in the incident light beam to cause the measurement ratio to reach or exceed the predetermined ratio, so that the instrument is calibrated and then removes light from the particles to be analyzed. The method includes being assayed to obtain a signal. In a preferred embodiment of this aspect of the invention, the method is used to calibrate a flow cytometry instrument, in which assay particles having diagnostic properties associated with the particles to be tested are run in flowing liquid water, and each assay particle is detecting both the optical signal and the noise signal associated with the assay particles, including passing the incident light beam substantially one at a time, and determining the signal to noise ratio. This ratio is recorded as the measured separation between the optical signal and the noise signal. The measured separation value is compared to a predetermined separation value that represents a threshold for critical performance of the instrument. If the predetermined separation value is not reached, the operation of the instrument is adjusted to bring the measured separation value to the predetermined separation value, thereby calibrating the instrument to obtain an optical signal from the particle that is subsequently analyzed. Another aspect of the invention is a reagent kit for use in calibrating an instrument for performing two-color fluorescence analysis of particles. The kit consists of a first container having assay particles capable of emitting fluorescence at a first wavelength. The second container contains assay particles capable of emitting fluorescence at a second wavelength. The third container contains assay particles that are substantially non-fluorescent. This kit or reagent is suitable for use in the above-described method for assaying flow cytometry instruments. Yet another aspect of the invention relates to a particle standard for calibrating fluorometric instruments, the standard being a tetroxide that does not emit any fluorescence, including autofluorescence, until it reacts with a specific fluorophore. Consists of osmium-fixed chicken red blood cells. According to the principles of the present invention, the assay methods and materials provide a significant improvement to currently known and used assay techniques. Enables an operator to calibrate a particle analysis device using stored threshold values. Preferably, the threshold value for specifying the critical performance of the device is displayed to the person performing the test procedure duration so that the test target is known. By adjusting and tuning the instrument until the threshold is achieved, the operator ensures that the instrument functions correctly for the test being performed. Techniques that rely on sense and vision of the test are
The assay procedure of the present invention reduces or eliminates the need for calibration errors and increases test reproducibility. Especially for flow cytometry instruments, as it compensates for spectral mixing through the implementation of the technique and provides information to the operator about the sensitivity of the instrument, in order to optimize the performance of the instrument, especially for the immunofluorescence analysis of particles. It should be pointed out that the advantages and features of the present invention extend beyond flow cytometry and can also be used in other analytical instruments such as automatic microscopes, fluorescence microscopes, quantitative microscopes, image analyzers, etc.

[Brief explanation of the drawing]

FIG. 1(1) to FIG. 1(d) are graphical representations of histograms illustrating counts for four parameter channels: volume, side scatter, fluorescent color 1, and fluorescent color 2, respectively; FIG. 1 further illustrates the volumetric results associated with noise cases and average channel statistics; the mean channel scale for side scatter, fluorescent color 1, and fluorescent color 2 also illustrates signal, noise, separation, and minima. . FIG. 2(j) is a point distribution graphical representation of two-color fluorescence analysis (F L 2 vs. FLI); FIG. 2(b) is a point distribution graphical representation of side scattering for fluorescence analysis; FIG. also displays channel averages for instrument array purposes. FIG. 3(a) is a point distribution graphical representation of two-color fluorescence analysis (PL 2 vs. FLI); FIG. 3(b) is a point distribution graphical representation of side scattering for volumetric analysis; Furthermore, the channel average of the distribution of fluorescence color 1 (FLl), fluorescence color 2 (FL2), and side scattering (SSC) by the photomultiplier tube of the instrument is displayed. Figure 4(a) is a point distribution graphical representation of a two-color fluorescence analysis (FL2 vs. FLL) illustrating uncorrected fluorescence analysis; Figure 4(a) shows the uncolored and colored distributions with fluorescent color 1. It also displays the mean channel difference between the
Figure (b) is a point distribution graph representation of two-color fluorescence analysis (F L 2 vs. FLI) illustrating the corrected fluorescence analysis; Figure 4 (b) shows the uncolored distribution and fluorescent color after correction. 2 distribution (FL2) is also displayed. Although the invention may be satisfied with embodiments in many different forms, preferred embodiments of the invention are herein described in detail with reference to the drawings. Examples herein are representative of the principles of the invention. It goes without saying that the invention is not intended to be limited to the illustrated embodiments.The scope of the invention is determined by the claims and their equivalents. Before referring directly to the drawings, the assay techniques described below in conjunction with the drawings are as they relate to flow cytometry equipment. A typical test procedure specifically described is:
Beckon Dickinson Immunocytometry System (BeckonD 1ckinson I ma+
(unocytometry systems),
Mountain Vie, :L
w), FAC3T marketed by Calipornia
N Associated with the analyzer. The histogram in Figure 1 and the point distribution displays in Figures 2-4 are typical displays seen on the screen of a FAC8 analyzer during an assay operation. can be supplied to flow cytometry equipment to facilitate various σ assay operations and functions. Briefly, the flow cytometry instrument to be assayed includes a nozzle or similar device for providing a liquid stream of particles to be analyzed, typically by covering the particles with a sheath fluid. , hydrodynamically converged,
Particle flow allows the liquid stream to flow through an incident light beam that is typically directed at the correct angle. As these particles pass through the light beam, optical characteristics associated with each particle can be detected. Thus, one or more photodetectors for measuring fluorescence, photodetectors for measuring absorption of light scattering in one or more directions, etc. may be included. These photodetectors contain a photomultiplier tube (PMT) that converts the optical signal into an electrical signal.In addition to detecting the light associated with the particle, these photodetectors also generate an electrical signal when the particle passes through an orifice. Impedance measurements can be made. This impedance measurement is related to particle volume and is based on the well-known Coulter
(Coulter) principle. The FACS analyzer performs the test as described below.
A PMT is equipped to detect two types of color: fluorescence and light scattering (in this case side scattering, which measures the scattering from the particle to approximately 9G''' relative to the incident light beam).FAC
The S analyzer also allows measurement of electrical impedance with particle volume. Class 4a parameters or properties of particles can thus be detected or measured, but all such parameters must be verified before the instrument is used in measuring these properties of particles in a sample. The verification technique ≠ of the present invention notifies the person measuring the device that the device will be tested based on the lower limit threshold of device performance. This minimum threshold tells us the sensitivity of the instrument for performing particle analysis, especially immunofluorescence measurements. For example, in addition to side scatter and volume, the particular flow cytometry instrument described has two colors of fluorescence, i.e. In total, 41!1 types of parameters can be detected. For the sake of explanation, using a typical application as an example, the first fluorescent color of the particles being analyzed is F
The second fluorescent color of the analyzed particles is the green spectral region associated with fluorescein-labeled particles, represented by ITC-stained cells, and the red color associated with phycoerythrin-labeled particles, represented by PE-stained cells. Of course, other fluorophores and labels may be used in the spectral range, if desired, depending on the test being performed and the particles or cells being analyzed. When the operator switches on the instrument in preparation for a prescribed test, for example at the beginning of the day, the optical elements of the instrument may not be aligned. If the tests being performed involve data acquisition for testing immune system particles, such as white blood cell samples, the operator should check whether the equipment has been calibrated to an appropriate sensitivity to perform these tests. The sensitivity performance of the instrument and the results it provides are shown in Figure 1. It goes without saying that this is a confirmatory test to determine whether the assay is suitable for analyzing certain types of particles, such as Particles. If the results of the sensitivity test meet the threshold for minimum performance of the instrument, it means that the instrument has been properly qualified and the instrument can then be used for particle analysis without further adjustment or alignment. There is no need to do or tune. However, for reliable measurements, it is usually necessary to calibrate the instrument with some further adjustment, so before determining the sensitivity with respect to the display in Figure 1, the operator should check the following
In the following discussion, where one may wish to ensure the adjustment and validation process described with respect to FIG. 4, the determination of sensitivity and how to achieve it will first be described based on the representation of FIG. To begin the procedure, the operator uses assay particles of the invention. A reagent kit is supplied that includes three containers, each container preferably having a different plastic microsphere or bead. In preferred form, these various beads are prepared in phosphate M buffer (PBS) containing gelatin, 0.1% Tween 20, and 0.1% sodium azide as a preservative.
). Approximately 5 micron diameter fluorescein labeled (FITC)
) Prepare the beads in the first container. Phycoerythrin I [31i (PE) beads with a diameter of approximately 5 microns are provided in a second container. Unlabeled beads approximately 4.5 microns in diameter are included in a third container. The concentration of beads in each of the three containers is approximately 106 particles/ml. Add three types of reagents, FITC, PE, and unlabeled beads, one drop at a time to sterile PBS3i+Il at a ratio of 1:1:1.
Prepare a sample tube by mixing in the following ratio. This sample tube containing the mixture is then placed in a FAC3 analyzer and measured in a known manner by passing the incident light beam substantially one particle at a time. In addition to detecting signals for volume, light scattering and two types of fluorescence determinants, noise signals for each parameter are also detected by the instrument. FIG. 1 represents the display on the instrument screen of four parameters in the form of a histogram, where the Y-axis of the histogram is the number of particle counts and the X-axis is the number of electrical channels during data acquisition. In the described implementation, there are 255 channels along the X-axis of the various histograms. Although histograms may be recorded or displayed as a logarithmic correlation so that the separation of the histogram peaks between noise and signal can be seen as a constant difference, in practice it is preferably expressed as a linear ratio. More specifically, the light-related signals are shown in FIGS. 1(b), (e) and (d) for side scatter, FITC and PE, respectively.
) is shown. Looking first at FIG. 1(b), the side scatter signal is shown by curve 12 and the noise is shown by region 14. Similarly, in FIG. 1(e), the FITC signal is shown by curve 16 and the noise is shown by region 18. In FIG. 1(d) the PE signal is shown by curve 20 and the noise is shown within region 22. For the volume shown in FIG. 1 (,L), the volume signal is shown by curve 24 and the noise detection is shown below the histogram as the number of noise instances. Scattered and fluorescent light-related signals, i.e., FIG. 1(b),
Regarding (a) and (d), it can be seen that there is a separation or difference between the actual optical signal and the noise signal. As mentioned above, the separation between signal and noise is preferably calculated on a logarithmic scale, but the ratio of signal to noise is represented as a linear difference on the X-axis of the histogram. Therefore, when looking at the histogram, the spacing and separation between signal and noise peaks with respect to scattering, FITC and PE can be easily recognized. In the histograms in Figures 1(c) and (d) for fluorescence analysis, what is actually being measured is the separation between the mean fluorescence intensities of labeled beads compared to unlabeled beads as provided by the reagents described above. It is. Therefore, in FIG. 1(e), curve 16 represents the average fluorescence intensity of FITC-labeled beads and curve 18 represents the resulting noise intensity of unlabeled beads in the reagent mixture used for assay purposes. . Similarly, in FIG. 1(d), curve 20 represents the average fluorescence intensity of PE-labeled beads and curve 22 represents the average intensity of the noise from unlabeled beads contributed by the reagent mixture for the assay. As such, by providing a single reagent mixture with equal proportions of FITC-labeled beads, PE-labeled beads, and unlabeled beads, calibration of the instrument to two-color fluorescence analysis can be easily accomplished. For side-scattered signals as represented in FIG. 1(b), the noise can actually be detected and the separation between the noise and the signal can be measured. This is necessary because the noise represented by region 14 in FIG. 1(b) is not due to unlabeled cells as would occur in the fluorescent signal. In this figure, side scatter (SSC) noise is induced with an unrelated signal, creating a reference signal for the noise. Therefore, the FL2 signal has been arbitrarily selected to provide a reference signal. Adjust the F'L2 standard on the flow cytometry instrument,
The case velocity (or flow rate of particles) through the equipment is defined as the volume, FL
Noise is introduced into the side scatter signal by approximately doubling the case rate when obtaining data for I and FL2. By this means noise is introduced into the side scatter signal and the amount of linearized separation between the noise and the scatter signal can be determined. In addition to the graphical display, which is preferably in the form of a histogram, the screen of the instrument being calibrated can also be programmed to display the results in the form of a digital readout. The software for the instrument, including the assay procedures, can be easily programmed to provide information regarding the accumulated data. Therefore, in FIG. 1, below the histogram, the volumetric results are recorded in digital readout format so that the noise cases and the average channel signal can be read by the measurer. For calibration of instruments such as the FAC3 analyzer, the volume average channel should be approximately mid-scale on the volume histogram. Reading the center scale indicates that the orifice through which the particles flow is correctly selected and the instrument volume standard is properly set; a noise instance level below 500 means that the instrument is properly set. do. The screen of FIG. 1 also displays digital readings associated with each of the histograms of FIGS. 1(b), (e), and (d). Various parameters of SSC, FLI and FL2 are displayed along with readings of signal, noise, separation, minimum and lot ID.Lot ID and minimum values are correlated to the mixture of assay beads on the instrument. Before entering the lot I into the program that defines the verification procedure.
Enter the D number. Because assay beads have variable properties, including varying fluorescence intensities, intensities, etc., instrument calibration must take these variable properties into account; therefore, using assay beads with lot ID numbers of Depending on the situation, various separation values are pre-established as minimum or dark values for minimum equipment performance. The software can be pre-programmed so that a lot ID number need only be entered into the program by the operator at the beginning of the assay procedure and a predetermined minimum separation value will be established and displayed on the display of FIG. The average median channel values for side scatter, FLI and PL2 signal and noise associated with the respective histograms of FIGS. 1(b), (c) and (d) are displayed at the bottom of FIG. 1. For example, The side scatter signal is 192
is shown to have an average channel median of -, whereas the average channel median of the noise is 16. The separation between these signal and noise results is recorded as 176. The minimum separation value previously determined for the side scatter signal is 160. This minimum side scatter separation value is the target level for the instrument to calibrate sufficiently to make side scatter measurements reliably. Therefore, the actual measured separation is 176, which exceeds the minimum. The instrument is therefore considered capable of validating subsequent particle tests using side scatter as the detection signal. Digital representations of average channel median values of signal, noise and separation are also provided for the FLI and PL2 signals. As shown in the example of FIG. Since both FLl and FL2 separation values exceed the minimum separation value, the operator can easily determine that the instrument is properly calibrated for these two types of signals as well. The software for the test procedure described above tests the signal-to-noise ratio on a logarithmic scale, but the peaks are far apart from each other on the screen, and therefore do not provide linearity (constant) spacing for convenient interpretation. Display the ratio based on. If much or all of the scattering or fluorescence light-related signals are measured with separation values below the predetermined minimum, further instrument adjustments must be made for proper assays. Figure 4 shows the FAC8AC8 analyzer performing this calibration adjustment to align the optical elements to optimize instrument performance, performing PMT37m, and correcting for spectral mixing and overlap in two-color fluorescence analysis of particles. Illustrate the display. F! equipment to ensure optical elements are properly aligned for optimal performance! To prepare, the assay can be performed using FITC-labeled beads from the first container of the reagent kit described above. FITC in sterile PBS1jf
Prepare a test tube for the FITC-labeled bead suspension by adding one drop of bead reagent. Next, attach this test tube to the FAC3 analyzer, and when combined with the assay software, the screen will appear as shown in Figure 2. A point distribution map will be displayed. Figure 2(a)
shows FL2 vs. FL1, and Figure 2(b) shows side scattering (S
FLI is below the 0 point distribution map showing SO) vs. FL1. Digital information regarding the channel averages of the FL2 and SSC signals is displayed. First of all, referring to Fig. 2(a), when the point distribution of green (FITC) beads is located near the center of the display, the instrument's fluorescent PMT FLI and FL2 displayed below the zero point distribution map for adjusting the voltage
In terms of signal averages, the operator ensures the test by maximizing these signals. The optical elements of the instrument, such as the tA optical lens control, should be adjusted so that the average of these signals is maximized, and yet they are maximized at about the same time. Near the maximum, the point distribution displayed in FIG. 2(a) is made by the measurer to have as few gaps as possible. Regarding the SSC signal average shown in FIG. 2(b) and below the point distribution diagram, a predetermined target value is usually the target for the verification. For example, the FAC8AC8 analyzer SSC signal channel average target is approximately 190. To achieve this target value, the operator adjusts the side scatter (SSC) PMT voltage of the instrument until the SSC signal average reaches approximately 190. Other adjustments to the optical elements or PMT may also be necessary to bring the SSC signal average to the target level. Both the zero point distribution and the number of digital readings help the operator calibrate the instrument to the appropriate optical alignment. do. Similar to the optical array, the photomultiplier tube (PMT) can also be adjusted during the assay procedure. FIG. 3 shows a typical point distribution diagram associated with PMT adjustment. Figure 3 (a) shows FL2 vs. F.
This is a point distribution diagram of LI, and Figure 3(b) is a point distribution diagram of LI.
C) Point distribution diagram of volume vs. volume. To decide whether to perform PMT714*, the operator performs the procedure using different assay particles than those described above. Add 1 drop of unlabeled beads from the container and
Prepare a non-labeled bead suspension using this non-F! When a test tube containing A-identified beads is attached to the FAC3 analyzer and combined with the assay software, a point distribution map as shown in Figure 3 is displayed on one screen.In addition to the 0 point distribution map, a third The figure is F
Three numbers representing the channel median mean values of the distributions of the three parameters LI, PL2 and ssc are also displayed. l! of the test particles at a controlled and constant speed.
While flowing through the instrument, the measurer adjusts the values of the FLI average, PL2 average, and SSC average to predetermined target values. For both FLl and FL2 averages, a predetermined target is set, for example around 30, and if that value is not achieved, the measurer will be required to adjust the FLI or F
Adjust the PMT voltage for each Fluorescence 1 or Fluorescence 2 on the instrument until the L2 channel average is set as close to 30 as possible. Similarly, the target for the SSC average is set to 190, for example. If this level is not initially displayed, the operator should adjust the PM of the light scattering of the instrument until the channel average of the SSC displayed on the screen is set as close to 190 as possible.
Adjust the T voltage. Of course, these average values represent typical target values and may vary depending on equipment design, testing performed, and other factors. If this cannot be achieved, the operator must return to the instrument arrangement procedure described in connection with FIG. After adjusting the PMT and aligning the optical elements of the instrument, one more adjustment is made to achieve instrument qualification.
This additional adjustment relates to correcting for spectral mixing and overlap when performing two-color fluorescence analysis. This procedure removes unwanted green signals from the red (PE colored) population and removes unwanted red signals from the red (FITC colored) population.
removed from the population and adjusted for fluorescence correction. To carry out the fluorescence correction procedure, it is preferable to start with a test tube of calibrator particles similar to that made for the sensitivity test described above, which is filled with F from the container described above in sterile PBS3at.
A 1:1:1 mixture of the three reagents is prepared by adding one drop each of ITC, PE and uncolored beads. When this test tube is attached to the analytical instrument, it is combined with the software program for the assay procedure and the screen shown in Figure 4 (
A point distribution map representing uncorrected fluorescence as seen in a) is immediately displayed. The three discrete clusters of dots displayed in Figure 4(a) represent unpigmented, green-pigmented and red-pigmented assay beads; It is typically located on the inside. Red (PE colored) beads are typically located above the non-colored population, shown as region 30. The green population is located to the right of the non-colored population, shown as region 32. The positions of regions 28, 30 and 32 are only approximate until fluorescence correction is provided by the instrument. Below the point distribution diagram in FIG. 4(a), there are digital displays of FL1 correction and PL2 correction. If these digital values do not vary as zero or near zero, adjustments for fluorescence correction must be made. until the correction value recorded on the screen is as close to zero as possible.
Correct for two-color fluorescence analysis by adjusting the instrument's FLI and FL2 corrections. After making adjustments, the correction value should vary and change equally above and below zero. For FL1 correction, the corrected value represents the difference between the average horizontal channel of the unpigmented population and the average horizontal channel of the green pigmented population, and the FLI correction adjustment is adjusted on the instrument until the value on the screen is zero. As it approaches, the green population 32 of the point map should move downward until it is equal to the uncolored population 28 about the horizontal axis parallel to the horizontal axis, as seen in FIG. 4(b). In a similar manner, the average channel difference listed next to the on-screen FL2 correction represents one difference in the average vertical channel of the uncolored and red colored populations. When the FL2 correction adjustment is adjusted to WA with the device and the value on the screen approaches zero, the red group 30 in the point distribution map becomes non-linear with respect to the vertical axis parallel to the vertical axis, as shown in Figure 4(b). It should move vertically until it is equal to the colored mass 28. FL
These adjustments of the I and FL2W nodes accomplish fluorescence correction by electrically subtracting the unwanted green signal population from the red population and electrically subtracting the unwanted red signal population from the green population. Correction is achieved when a predetermined value of on-screen fluorescence correction, which is set to zero by the computer program, is met. If a satisfactory or target setting cannot be achieved, the operator must return to the alignment and PMT adjustment procedure as described above. Once alignment, PMT adjustments and fluorescence corrections are achieved, the operator returns to the sensitivity test described above based on FIG. If a minimum separation value is achieved for each parameter being calibrated, the operator can proceed to use the instrument with sufficient confidence that the test being performed has been properly calibrated. Regarding the assay particles utilized in the assay procedures described herein, a variety of particles can be used as standards, e.g. particles that have, or can be treated to have, properties similar to the actual particles undergoing analysis. Alternatively, biological samples such as cells can be used. Along this line, chicken red blood cells can be used with appropriate selection techniques. Particles in a modified form of chicken red blood cells can be used as particle standards. In particular, fluorescently labeled osmium tetroxide-fixed chicken red blood cells have scattering properties similar to those of biological cells, making them useful as particle standards; These cells are biotinylated and reacted with a fluorophore coupled to avidin using known methods. The fluorescence of avidin complexes bound to cells is not quenched by osmium tetroxide on the cells. The specific fluorophores bound to avidin have a distinctive spectral character in cell surface coloration. Many types of fluorophores can be used in the conjugate, including: The most suitable assay particles for the present invention are plastic microspherical beads. Such beads may be of generally uniform diameter and have suitable surface characteristics for surface attachment of fluorophores and other dye labels. Substantially spherical beads are fabricated with diameters between 0.5 and 20 microns (most preferably between 2 and 8 microns) to make these assay beads similar in size to white blood cells. do. Beads are usually produced as solid bodies, but they can also be hollow or vesicular, such as ribosomes or other microcarriers. Furthermore, the beads do not have to be perfectly spherical to work in accordance with the present invention; polystyrene, polyacrylamide and other latex materials can be used to form the beads;
Other plastic materials such as polyvinyl chloride, polypropylene, etc. can also be utilized. In addition to the fluorescein and phycoerythrin labeling on beads described above,
Allophycocyanin, Texas Red, rhodamine, rhodamine-type compounds and other fluorescent dyes can also be used as fluorescent labeling agents for the present invention. In making kits of these reagents for zero-instrument assays, labeled or unlabeled beads are Preferably, the beads are provided as a dispersion in water so that they are present in a concentration ranging from 105 to 10 particles/ml (preferably at least 10' particles/ml). Although the assay procedure has been described above using a flow cytometry device as a specific example, the present invention is not limited thereto.In fact, the assay procedure of the present invention can be applied to instruments that analyze not only moving particles but also non-moving particles. Various microscopes such as fluorescence, automatic and fixed Ji' Accordingly, the present invention provides methods and materials for calibrating, prior to use, an instrument for obtaining at least one light-related signal from particles under analysis. Using appropriate software, the present invention can indicate the minimum signal-to-noise ratio that must be met in order to decide to calibrate a particular light-related parameter with the instrument. By recording the ratio of signal to noise as a difference on a linearity scale, reliance on eye measurements and errors are substantially reduced or eliminated, while providing convenience to the operator. The techniques and materials of the present invention also provide great reliability for clinical diagnostic applications.

[Brief explanation of the drawing]

FIGS. 1(a) to d) are histograms showing the relationship between particle volume, side scattering, fluorescent color 1 and fluorescent color 2, and particle count number, respectively.
) is an example of the information that is displayed together with the above-mentioned stograph on the display device of the device in order to certify the device according to the present invention in combination with the above-mentioned stograph. Figure 2 (a) is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2 in two-color fluorescence analysis, and Figure 2 (b) is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2 in two-color fluorescence analysis.
It is a point distribution graph showing the relationship between and side scattering, and Figure 2 (
The letters at the bottom of a) and (b) illustrate a state in which an example of the information necessary to certify the device according to the present invention using the above point distribution graph is displayed on the display section of the device. be. FIG. 3(a) is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2 in two-color fluorescence analysis, and FIG. 3(b) is a point distribution graph showing the relationship between side scattering and particle volume distribution. The letters under FIGS. 3(a) and 3(b) indicate the state in which an example of the information necessary to certify the device according to the present invention using the above-mentioned point distribution graph is displayed on the display section of the device. There are illustrations. FIG. 4(a) is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2 in fluorescence analysis with an uncorrected instrument, and FIG. 4(b) is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2 in fluorescence analysis with an instrument after correction. FIG. 4 is a point distribution graph showing the relationship between fluorescence 1 and fluorescence 2.
The characters below a) and (b) illustrate a state in which an example of information useful for utilizing the above-mentioned point distribution graph is displayed on the display section of the device. (a) (b) (c)
(d)(a)
(b) FLI average: 32 FL2
Kii7:31 Kiyoshi:189FL I
FL1(a)
(b) Day J 1st penalty, 114 Sac41% + average: 190 Sleeves - 2nd correction = 39 Sleeves - 2nd correction: 94 Seds Sho LI

Claims (9)

[Claims] (1) A first container having assay particles capable of emitting fluorescence at a first wavelength; a second container having assay particles capable of emitting fluorescence at a second wavelength; and a third container having assay particles substantially unable to emit fluorescence. ; A reagent kit used for assaying an instrument that performs two-color fluorescence analysis of particles consisting of; 2. The kit of claim 1, wherein all of the assay particles are spherical beads having a diameter between substantially 0.5 and 20 microns. (3) The kit according to claim 2, wherein the beads are made of a plastic material. (4) The kit according to claim 1, wherein the particles in the first container are labeled with a surface-bound fluorescent stain. (5) The kit according to claim 4, wherein the fluorescent stain is fluorescein. (6) The kit according to claim 1, wherein the particles in the second container are labeled with a surface-bound fluorescent stain. (7) The kit according to claim 6, wherein the fluorescent stain is phycoerythrin. (8) The kit of claim 1, wherein the particles in each of the three containers are present in the aqueous solution at a concentration of at least 10^5 particles/ml. (9) a first container having beads that are labeled with fluorescein and are substantially spherical; a second container that has beads that are labeled with phycoerythrin and are substantially spherical; third with beads
The beads in all the above containers are made of plastic, have a diameter between 0.5 and 20 microns, and are present in an aqueous solution in said container at a concentration of at least 10^5 beads/ml. A reagent kit used to calibrate an instrument for performing two-color fluorescence analysis of particles, comprising: